The invention relates to the selection of ions of a predetermined range of mobilities, preferably for being analyzed by mass spectrometry. Mass spectrometers can only ever determine the ratio of the ion mass to the charge of the ion. Where the terms “mass of an ion” or “ion mass” are used below for simplification, they always refer to the ratio of the mass m to the dimensionless number of elementary charges z of the ion. This charge-related mass m/z has the physical dimension of a mass, but it is often also called “mass-to-charge ratio”, although this is incorrect with regard to the physical dimension. The term “ion species” shall denote ions having the same chemical formula, the same charge and the same three-dimensional structure. Ion species generally comprise all ions of an isotope pattern containing ions of slightly different masses, but virtually the same mobilities.
Different kinds of isomers are known for bioorganic molecules: isomers related to the primary structure (structural isomers) and isomers related to the secondary or tertiary structure (conformational isomers). These isomers have different geometrical forms but exactly the same mass. It is therefore impossible to differentiate between them on the basis of their mass. Some information as to the structure can be obtained from fragment ion mass spectra; however, an efficient and certain way to recognize and distinguish such isomers is to separate their ions according to their different mobilities.
Nowadays, the mobility of ions is often measured via their drift velocities in a drift region under the influence of an homogeneous electric field along the drift region. The drift region is filled with an inert, stationary gas (such as helium, nitrogen or argon). The ions of the substance under investigation are pulled through the gas by means of the electric field, which is produced by suitable DC potentials applied to ring electrodes arranged along the drift region. The large number of collisions with the gas molecules results in a constant drift velocity vd for each ion species which is proportional to the electric field strength E: vd=μ×E. The proportionality factor μ is called the “ion mobility” of the ion species. The ion mobility μ is a function of the gas temperature, gas pressure, type of gas, ion charge and, in particular, the collision cross-section of the ions.
Isomeric ions with the same charge-related mass m/z but different collision cross-section have different ion mobilities in a gas of the same temperature, pressure and type. The Isomer with the smallest geometric dimension exhibits the greatest mobility compared to other isomers and therefore the highest drift velocity through the gas. Unfolded protein ions undergo more collisions than tightly folded proteins. Protein ions which are unfolded or partially folded therefore arrive at the end of the cell later than strongly folded ions of the same mass. Structural isomers, for example proteins with glycosyl, lipid or phosphoryl groups at different sites, also have different collision cross-sections, which allow them to be distinguished by measuring their ion mobility.
In chemical and biological research, it has become more and more important to have knowledge about the folding structures of bioorganic ions, which can be identified via their mobility. Therefore devices to measure the mobility of ions have been incorporated into mass spectrometers, in particular, in order to combine the measurements of the charge-related mass of ions with the measurement of collision cross-sections. The folding structures determine the mechanism of action and thus the function of the molecules in the living organism; different foldings can signify normal or abnormal functioning of biopolymers in biosystems, and hence health or disease of tissue parts or even whole organisms.
A number of academic research groups have coupled ion mobility spectrometers with mass spectrometers. A pressure in the range of several hectopascals has been adopted almost universally in the drift region; the drift region for higher mobility resolutions measures up to four meters and more, and electric field strengths of 2,000 volts per meter and more are applied. In this pressure range, the drifting ions appear to form hardly any complexes with other substances, so the mobilities of the ion species can be measured without interferences, unlike mobility measurements at atmospheric pressure. But in the long drift regions, the ions also diffuse radially over long distances, and therefore quite large diameters have to be chosen for these drift regions.
The ions are usually introduced into the drift region by pulsing a shutter grid at the entrance of the drift region to form ion clouds, having the shape of thin slices, which are pulled through the drift region by the electric field. In the gas of the drift region, these ion clouds are subject to diffusion, caused by collisions statistically distributed in terms of spatial directions and kinetic energies due to the molecular Brownian motion. The diffusion takes place in all directions from the cloud, thus also in radial direction to the drift direction. The gas in the drift region is sometimes kept at temperatures of between about 150 and 300 degrees Celsius, but can also be cooled down for special experiments.
The resolving power of a ion mobility spectrometer is defined as Rmob=μ/Δμ=vd/Δvd, where Δμ is the width of the ion signal of the mobility μ at half height, measured in units of the mobility, and Δvd is the correspondent difference in drift velocity Vd. The resolving power Rmob is influenced predominantly by the diffusion broadening of the ion clouds, particularly for long drift regions and high electric field strengths; all other influences, such as the space charge, tend to be negligibly small.
The part of the resolving power determined by the diffusion broadening is given by the equation
            R      d        =                            zeEL          d                          kT          ⁢                                          ⁢          ln          ⁢                                          ⁢          2                      ,where z is the number of unbalanced elementary charges e of the ions, E the electric field strength, Ld the length of the drift region, k the Boltzmann constant and T the temperature of the gas in the drift region. A high mobility resolution can thus only be achieved by means of high field strengths E, long drift regions Ld, or low temperatures T. The part Rd of the resolving power that is given by the diffusion is not dependent on either the type or pressure of gas in the drift region; the mobility K0 itself, however, does depend not only on the temperature, but also on the pressure and type of gas.
Compared to the numerical values for mass resolutions in mass spectrometry, the mobility resolutions which can be achieved in practice are generally very low. The first commercial ion mobility spectrometer for bioorganic ions has a mobility resolution of only Rmob=40. With a mobility resolution of Rmob=40, two ion species whose collision cross-sections differ by only five percent can be well separated into two peaks.
Only highly specialized academic working groups have, as yet, been able to achieve significantly higher mobility resolutions than Rmob=100, in rare individual cases up to Rmob=200, with long drift lengths roughly between two and six meters and field strengths between 2,000 and 4,000 volts per meter, making it possible to differentiate between ion species whose mobilities differ by only one to three percent. Ion mobility spectrometers with a resolution above Rmob=100 shall be called “high resolution” here.
In long mobility drift regions, the transverse diffusion widens the ion clouds broadly. Therefore, longer drift regions must also have a large diameter so that the ions do not touch the enclosure of the drift region. A well established method is to guide the ions back to the axis of the drift region after they have passed through a part of the drift region, about two meters, for example. This is done using so-called “ion funnels”. These consist of a larger number of parallel ring diaphragms, spaced apart from each other in the order of millimeters. The inner diameter of the diaphragm's aperture taper continuously from the diameter of the drift region, 30 to 40 centimeters, for example, down to a few millimeters and thus form a funnel-shaped enclosed volume. The two phases of an RF voltage, usually of several megahertz and between a few tens of volts and one hundred volts, are applied alternately to the apertured diaphragms, thus generating a pseudopotential which keeps the ions away from the funnel wall. An axial DC voltage gradient is superimposed on the RF voltage generating a DC electric field along the funnel. This electric field pushes the ions slowly towards the narrow exit of the funnel and through it. The ion funnel does not measurably reduce the mobility resolution of a long drift region.
Ion funnels are not only used to guide the ions back to the axis of the drift regions in ion mobility spectrometers; they are also used in mass spectrometers in general to gather the ions from larger ion clouds and to thread these ions into narrow ion guides. Such ion funnels are often found in mass spectrometers with electrospray ion sources; the ions generated outside the vacuum system are transferred, together with a curtain gas, through inlet capillaries into the vacuum system, where they are captured by ion funnels and freed of most of the curtain gas. Some mass spectrometers even contain two such ion funnels, placed in series, in order to move the ions quickly from regions with higher pressure of several hectopascals at the end of the inlet capillary to regions with lower pressure of less than 10−2 pascal.
High-resolution time-of-flight mass spectrometers with orthogonal injection of the ions (OTOF-MS), in particular, have proven successful for combinations of mobility spectrometers with mass spectrometers. For such combinations, the common high-resolution ion mobility spectrometers of the drift type have the disadvantage of being several meters long. For the construction of small, high-resolution mobility analyzers, one therefore has to look for a solution which shortens the overall length but does not diminish the mobility resolution.
In document U.S. Pat. No. 7,838,826 B1 (M. A. Park, 2008), an ion mobility spectrometer is presented, the size of which amounts to about ten centimeters only. It is based upon a gas flow driving ions against and over an electric counter-field barrier inside a modified ion funnel of a time-of-flight mass spectrometer. Since the publication of this device, ion mobility resolutions in excess of Rmob=100 have already been achieved with this small spectrometer. Considerably higher resolutions can be expected by future improvements.
Ion mobility spectrometers with moving gases and electric barriers date back to the year 1898, when J. Zeleny published an article entitled “On the Ratio of the Velocities of the Two Ions produced in Gases by Rontgen Radiation; and on some Related Phenomena”, in Philosophical Magazine, No. 46, pp. 120-154. Zeleny generated ions between two parallel grids producing a homogeneous electric field and let a broad laminar flow of gas pass through the two parallel grids in a direction normal to the grids, counteracting the electric field produced between the grids. By changing the electric field, he could separate ions by their different mobilities. Since then, several patents and patent applications were published, using the principle of gas flows for the measurement of ion mobilities; in most cases, however, driving ions by electric fields of varying strengths against a moving gas. For none of these ion mobility spectrometers, however, resolutions near to Rmob=100 have been reported.
The apparatus of M. A. Park, as described in U.S. Pat. No. 7,838,826 B1, and the potential and field profiles for its operation are schematically illustrated in FIGS. 1A to 1D. FIG. 1B shows, how the parts (10) and (12) of a quadrupolar funnel, open to gas movement between the electrodes, are separated by a closed, tube-like quadrupole device (11) shown in FIG. 1A, which is vertically segmented into slices of thin electrodes (17, 18) arranged around an axis (denoted as the z-axis) with a circular central opening forming the tube. The electrodes are separated by insulating material closing the gaps. FIG. 1A shows the shape of the electrodes of the funnel (15, 16) in a direction normal to the z-axis and the shape of the electrodes that form quadrupole tube (17, 18) in a direction normal to the z-axis, the latter with equipotential lines of the quadrupolar field inside the tube. A differential pumping system of a mass spectrometer (not shown), surrounding the ion mobility spectrometer, is designed to cause a gas to flow through the tube of part (11) in a laminar way, so that the gas flow assumes the usual parabolic velocity profile (14). Ions, which enter the first part (10) of the funnel together with the gas, are collisionally focused into the axis of the tube.
FIGS. 1C and 1D show different DC potential profiles (22 to 26) along the z-axis of the tube, and corresponding barriers Ez of the electric counter field, respectively. The operation of the ion mobility spectrometer will be described by the sequence in which the DC potential profiles are supplied. The operation starts with a filling process. The steepest potential profile (22) is generated, producing the highest electric field barrier, collecting ions of all ion mobilities. The ions (27) are blown by the gas flow against the field barrier and are stopped there because they cannot surmount the field barrier. Ions with high mobility gather at the foot of the barrier, ions with low mobility gather near the summit, as symbolically indicated by the smaller and larger cross sections of the ions (27). When a suitable number of ions have been collected, the supply of further ions is stopped; for instance, by reversing the direction of the DC field within the ion funnel (10). Then, to acquire a spectrum, the potential profile (22) is lowered in height continuously in a scan (28), through potential profiles (23) to (26), resulting in a decrease of the electric barrier. During the scan, ions of higher and higher mobilities (smaller and smaller cross sections) can surmount the decreasing summit of the barrier, exit the spectrometer and be measured by an ion detector, favorably by a mass spectrometer. The measured ion current curve reflects directly the ion mobility spectrum. This device is denominated a “TIMS”, or “trapped ion mobility spectrometer”.
With this instrument, ion mobility resolution Rmob increases with increasing pressure, at least up to a few hectopascal, with increasing gas flow plus barrier height, and with decreasing scan speed. The device turns out to be at least as good as drift tubes of about one to two meters in length with stationary gas as described above.
Because only a moderate amount of ions is trapped in each analyzing cycle, only a limited number of ions of each mobility is available in each single scan, in most cases not enough for a thorough investigation of ions of a selected mobility in a mass spectrometer, for instance, by the generation of fragment ion spectra. There is still a need to collect more ions of a selected mobility, or to produce a constant current of ions with a selected mobility, for example by a mobility filter.
It should be mentioned here, that there are other types of short ion mobility spectrometers using gas flows. Document US 2010/0,090,102 A1 (O. Raether et al, 2008) describes, how a freely expanding gas flow from a small opening can be used to drive entrained ions over an electrical barrier within an ion funnel. In document GB 2473723 A (J. Franzen, 2009), an apparatus is presented which generates a supersonic gas flow by a Laval nozzle, the supersonic gas flow driving entrained ions over an electrical barrier. In this case, the supersonic gas flow with entrained ions is not enclosed by any radially confining field, particularly not by an RF multipole field.